1. Technical Field
The present disclosure relates to methods for the quantification of biological material in a sample, and to devices for partitioning and holding the biological material during quantification.
2. Discussion of Related Art
The determination and enumeration of microbial concentration is an essential part of microbiological analyses in many industries, including water, food, cosmetic, and pharmaceutical industries. The classical methods of detection and quantification of biological material are performed using semi-solid nutrient agar medium (e.g. pour plate method, membrane filtration) or liquid nutrient medium (e.g. the most probable number method). If a pour plate method is being performed, the sample being tested for microbial contamination is first dispensed in a Petri-dish. Then 15 ml of the appropriate nutrient medium is poured over the sample. The Petri-dish is then left to solidify at room temperature for approximately 20 minutes and then incubated at a specific temperature for a specific time, and any resulting colonies are counted. Drawbacks for the pour plate method include bacterial colonies, which may be too small or overlapping each other for counting and particulate matter in the samples, which may also interfere with counting. For the membrane filtration method, the required volume of sample is filtered through a membrane of a very small pore size to non-specifically trap bacteria. The membrane is then placed on a prepared solid medium, which supports the growth of the target bacteria. The medium is then incubated at a specific temperature for a specific time, and any resulting colonies are counted. Drawbacks of membrane filtration include particulate matter other than bacteria in the sample (e.g., a waste water sample) may clog the membrane making it unusable and bacterial colonies may be too small or overlapping each other making it difficult to count.
Improved methods using solid-base nutrient medium to support microbial growth for microbial detection and quantification include READIGEL® (3M Microbiology Products, St. Paul, Minn.), which uses a special chemically treated Petri-dish. The sample is inoculated into a growth medium and poured into the plate. The sample/medium mixture is solidified 20 minutes after it comes into contact with the chemicals coated in the plates. Alternatively, PETRIFILM® (3M Microbiology Products, St. Paul, Minn.), which is an adhesive tape-like material having a coated media deposited thereon may also be used. This arrangement forms a thin layer of growth media that hydrolyzes and gels upon contact with liquid samples. A cover piece helps to disburse the sample inoculums and also acts as a cover for incubation. These methods offer improvement over the pour plate and membrane filtration methods in that these methods are easier to perform. However, these methods suffer the same limitations as those of pour plate and membrane filtration methods as described above.
The most probable number method (MPN) is well known and described, for example, in Recles et al., “Most Probable Number Techniques” published in “Compendium of Methods for the Microbiological Examination of Foods”, 3rd ed. 1992, at pages 105-199, and in Greenberg et al., “Standard Methods For the Examination of Water and Wastewater” (8th ed. 1992).
Microbial quantification devices and methods using the MPN method are commercially available. Devices and Methods such as Quanti-Tray® and Quanti-Tray® 2000 (IDEXX Corporation, Westbrook, Me.) are used for microbial quantification for drinking water, surface water, and waste water samples. A detailed disclosure of these tests may be found in Naqui et al. U.S. Pat. Nos. 5,518,892; 5,620,895; and 5,753,456. To perform these tests the separate steps of adding the sample/reagent to the device and then sealing the device with a separate sealing apparatus are required before the incubation period. These methods and devices offer a significant improvement over the traditional multiple tube fermentation techniques in terms of their ease of use and also allow for accurate quantification of microorganisms in the sample. However, devices of this type may require an instrument to distribute the sample/medium mixture into each individual compartment and are more applicable for enumerating microbial populations in the microaerophilic condition.
Croteau et al. also describe a method and device for quantification of biological material in a sample using the MPN method in U.S. Pat. Nos. 5,700,655; 5,985,594; and 6,287,797. The device uses a flat horizontal incubation plate and the surface is divided into a plurality of recessed wells. The liquefied sample/medium mixture is poured onto the surface of the device and after gentle mixing the sample/medium mixture is distributed into the recessed wells and held in the well by surface tension. The plate is then incubated at a specific temperature for a specific time until the presence or absence of the biological material is determined. Pierson et al. in U.S. Pat. No. 6,190,878, entitled “Methods and Devices for the Determination of Analyte in a Solution”, disclose devices using a flat horizontal surface, which is divided into a plurality of recessed wells. Others have one or more surfaces with reagent islands immobilized thereon. Each well or wells or reagent islands are sized and shaped, and formed of a suitable material to hold the aliquot within the well or reagent islands by surface tension. These devices offer improvement over the gel-based methods for microbial enumeration by providing the benefit of easy result interpretation and higher counting ranges. These methods and devices potentially may have some disadvantages. Sample inoculation may be hampered by air bubbles, which form in the wells during the inoculation of samples and requires a pipetting step.